Two cytosolic sources of NADPH, the hexose monophosphate pathway and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), have been found to be essential in preventing the accumulation of deleterious oxidative byproducts of endogenous metabolic pathways including peroxisomal beta-oxidation and mitochondrial respiration. Loss of glucose-6-phosphate dehydrogenase (ZWF1, the first enzyme in the hexose monophosphate pathway) and of IDP2 results in a rapid loss in viability of yeast cells transferred to medium with oleate or acetate as the carbon source. In contrast, loss of major cellular peroxidative enzymes has no effect on growth with these carbon sources. The lethality resulting from endogenous metabolic byproducts, and the specific requirement for cytosolic sources of NADPH to protect from this lethality, are novel observations in the area of cellular oxidative stress. Further investigation of these phenomena is proposed because oxidative damage to cellular macromolecules has been associated with numerous degenerative diseases and with the process of aging. Primary aims of this proposal include the following: (A) Proteins and DNA from strains lacking cytosolic sources of NADPH will be examined to identify specific cellular and organellar macromolecular targets of deleterious oxidants generated by normal metabolic pathways. (B) We will assess the importance of cytosolic sources and of levels of NADPH as determinants of longevity, using yeast strains lacking or overexpressing these key antioxidant enzymes for life-span analysis. (C) We will initiate biochemical analyses of mammalian IDP2, and examine the physiological relevance of co-localization of this enzyme in the cytosol and in peroxisomes using both yeast and mouse models. In addition, we will continue to examine the structural basis for the unique physiological functions of yeast IDP2. (D) Finally, we will assess global changes in pools of central metabolites and reducing equivalents to directly test fundamental hypotheses related to loss or replacement of cytosolic sources of NADPH.